Bottom Line:
During prolonged stimulation, the ratio of steady state vs. peak IPSC amplitude was significantly lower for glycinergic IPSCs.In the absence of receptor blockers, repetitive light stimulation was only able to effectively evoke IPSCs up to 20 Hz in both bushy and multipolar neurons.We conclude that local GABAergic release within the VCN can differentially influence bushy and multipolar cells.

Affiliation: Department of Otolaryngology/Head and Neck Surgery, University of North Carolina at Chapel Hill Chapel Hill, NC, USA.

ABSTRACTBoth glycine and GABA mediate inhibitory synaptic transmission in the ventral cochlear nucleus (VCN). In mice, the time course of glycinergic inhibition is slow in bushy cells and fast in multipolar (stellate) cells, and is proposed to contribute to the processing of temporal cues in both cell types. Much less is known about GABAergic synaptic transmission in this circuit. Electrical stimulation of the auditory nerve or the tuberculoventral pathway evokes little GABAergic synaptic current in brain slice preparations, and spontaneous GABAergic miniature synaptic currents occur infrequently. To investigate synaptic currents carried by GABA receptors in bushy and multipolar cells, we used transgenic mice in which channelrhodopsin-2 and EYFP is driven by the vesicular GABA transporter (VGAT-ChR2-EYFP) and is expressed in both GABAergic and glycinergic neurons. Light stimulation evoked action potentials in EYFP-expressing presynaptic cells, and evoked inhibitory postsynaptic potentials (IPSPs) in non-expressing bushy and planar multipolar cells. Less than 10% of the IPSP amplitude in bushy cells arose from GABAergic synapses, whereas 40% of the IPSP in multipolar neurons was GABAergic. In voltage clamp, glycinergic IPSCs were significantly slower in bushy neurons than in multipolar neurons, whereas there was little difference in the kinetics of the GABAergic IPSCs between two cell types. During prolonged stimulation, the ratio of steady state vs. peak IPSC amplitude was significantly lower for glycinergic IPSCs. Surprisingly, the reversal potentials of GABAergic IPSCs were negative to those of glycinergic IPSCs in both bushy and multipolar neurons. In the absence of receptor blockers, repetitive light stimulation was only able to effectively evoke IPSCs up to 20 Hz in both bushy and multipolar neurons. We conclude that local GABAergic release within the VCN can differentially influence bushy and multipolar cells.

Figure 7: Light evoked inhibiton only entrains for low frequencies. (A) Example IPSCs to light trains consisting of ten 2-ms pulses. Notice that IPSCs are only effectively evoked throughout the trains at 20 Hz in this neuron. At 50 and 100 Hz, light failed to evoke IPSCs in the later phase of the trains. (B) Example IPSCs to light trains in a multipolar neuron. (C) Normalized IPSC amplitudes through the light stimulus trains at different frequencies in bushy neurons. (D) Normalized IPSC amplitudes through the light stimulus trains at different frequencies in multipolar neurons. In (C, D), thin lines show individual neurons; thick lines are group averages for each frequency.

Mentions:
Under physiological conditions, many neurons in the the auditory system fire at relatively high rates. However, light evoked firing in inhibitory neurons in this VGAT-ChR2-EYFP mouse may not be able to follow high rates due to desensitization of ChR2 currents (Lin et al., 2009). We therefore tested the effectiveness of the light evoked inhibitory synaptic transmission with repeated stimulation. Ten light pulses of 1–2 ms duration were presented at frequencies of 10, 20, 50 and 100 Hz to drive inhbitory synaptic transmission onto bushy and multipolar neurons. Cells were held at −57 mV while using a Cs-based electrode solution with 38 mM chloride. No strychnine or GABAzine was used in this set of experiments, and excitatory transmission was blocked by including 5 µM CNQX in the bath. Under these conditions (brief light pulse stimulation), the IPSCs were only inward as the outward GABAergic IPSCs were masked by the larger glycinergic IPSCs (Figures 7A,B).

Figure 7: Light evoked inhibiton only entrains for low frequencies. (A) Example IPSCs to light trains consisting of ten 2-ms pulses. Notice that IPSCs are only effectively evoked throughout the trains at 20 Hz in this neuron. At 50 and 100 Hz, light failed to evoke IPSCs in the later phase of the trains. (B) Example IPSCs to light trains in a multipolar neuron. (C) Normalized IPSC amplitudes through the light stimulus trains at different frequencies in bushy neurons. (D) Normalized IPSC amplitudes through the light stimulus trains at different frequencies in multipolar neurons. In (C, D), thin lines show individual neurons; thick lines are group averages for each frequency.

Mentions:
Under physiological conditions, many neurons in the the auditory system fire at relatively high rates. However, light evoked firing in inhibitory neurons in this VGAT-ChR2-EYFP mouse may not be able to follow high rates due to desensitization of ChR2 currents (Lin et al., 2009). We therefore tested the effectiveness of the light evoked inhibitory synaptic transmission with repeated stimulation. Ten light pulses of 1–2 ms duration were presented at frequencies of 10, 20, 50 and 100 Hz to drive inhbitory synaptic transmission onto bushy and multipolar neurons. Cells were held at −57 mV while using a Cs-based electrode solution with 38 mM chloride. No strychnine or GABAzine was used in this set of experiments, and excitatory transmission was blocked by including 5 µM CNQX in the bath. Under these conditions (brief light pulse stimulation), the IPSCs were only inward as the outward GABAergic IPSCs were masked by the larger glycinergic IPSCs (Figures 7A,B).

Bottom Line:
During prolonged stimulation, the ratio of steady state vs. peak IPSC amplitude was significantly lower for glycinergic IPSCs.In the absence of receptor blockers, repetitive light stimulation was only able to effectively evoke IPSCs up to 20 Hz in both bushy and multipolar neurons.We conclude that local GABAergic release within the VCN can differentially influence bushy and multipolar cells.

Affiliation:
Department of Otolaryngology/Head and Neck Surgery, University of North Carolina at Chapel Hill Chapel Hill, NC, USA.

ABSTRACTBoth glycine and GABA mediate inhibitory synaptic transmission in the ventral cochlear nucleus (VCN). In mice, the time course of glycinergic inhibition is slow in bushy cells and fast in multipolar (stellate) cells, and is proposed to contribute to the processing of temporal cues in both cell types. Much less is known about GABAergic synaptic transmission in this circuit. Electrical stimulation of the auditory nerve or the tuberculoventral pathway evokes little GABAergic synaptic current in brain slice preparations, and spontaneous GABAergic miniature synaptic currents occur infrequently. To investigate synaptic currents carried by GABA receptors in bushy and multipolar cells, we used transgenic mice in which channelrhodopsin-2 and EYFP is driven by the vesicular GABA transporter (VGAT-ChR2-EYFP) and is expressed in both GABAergic and glycinergic neurons. Light stimulation evoked action potentials in EYFP-expressing presynaptic cells, and evoked inhibitory postsynaptic potentials (IPSPs) in non-expressing bushy and planar multipolar cells. Less than 10% of the IPSP amplitude in bushy cells arose from GABAergic synapses, whereas 40% of the IPSP in multipolar neurons was GABAergic. In voltage clamp, glycinergic IPSCs were significantly slower in bushy neurons than in multipolar neurons, whereas there was little difference in the kinetics of the GABAergic IPSCs between two cell types. During prolonged stimulation, the ratio of steady state vs. peak IPSC amplitude was significantly lower for glycinergic IPSCs. Surprisingly, the reversal potentials of GABAergic IPSCs were negative to those of glycinergic IPSCs in both bushy and multipolar neurons. In the absence of receptor blockers, repetitive light stimulation was only able to effectively evoke IPSCs up to 20 Hz in both bushy and multipolar neurons. We conclude that local GABAergic release within the VCN can differentially influence bushy and multipolar cells.